Polyglutamine expansion affects huntingtin conformation in multiple Huntington’s disease models

Abstract

Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington’s disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine- and temperature-dependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperature- and polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients.

N-terminal phosphorylation of Huntingtin: A molecular switch for regulating Htt aggregation, helical conformation, internalization and nuclear targeting

“…Our CD studies showed that phosphorylation at S13 and/or S16 led to complete disruption of Nt17 helix formation in the absence of presence of lipid vesicles and reversed the pT3-induce α-helix formation of Nt17. These findings are consistent with a recent study by Daldin et al demonstrating that phosphorylation of S13 and S16 (pS13/pS16-Q 42 ) conveys increased conformational flexibility in temperature and polyQ repeat lengthdependent experiments (65). They proposed that disruption of the Nt17 α-helix is responsible for this observation.…”

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

“…However, the effects of bona fide phosphorylation on mutant HTTex1 aggregation were evaluated only in vitro using semisynthetic proteins (Daldin et al, 2017;Deguire et al, 2018). Therefore, we tested whether TBK1-induced phosphorylation affected the aggregation of mutant HTT in HEK293 cells.…”

“…We recently showed that phosphorylation of HTT at S13 and/or S16 inhibits mutant HTTex1 aggregation in vitro (Deguire et al, 2018) and increases its conformational flexibility (Daldin et al, 2017). A direct comparison of the effect of phosphomimetics and bona fide phosphorylation at these sites also showed that the phosphomimetics (S13D/S16D) only partially reproduced the effect of phosphorylation on aggregation and did not reproduce the effect of single and double phosphorylation at these residues on the helical conformation of N17 (Deguire et al, 2018).…”

Proteostasis in Huntington's disease: disease mechanisms and therapeutic opportunities

“…The exon 1 structure is altered by polyQ expansion although there is no consensus on which morphology of exon 1 represents the toxic aggregate species. Whilst exon 1 HTT protein fragments have been shown to be a degradation product in many HD models and their aggregation underlies disease pathology [34,35] , larger fragments and full-length HTT are almost certainly involved in this oligomerisation process in human patients [36] with post-translational modification, particularly phosphorylation, of HTT playing a crucial role in aggregation and toxicity [37] . To date, the precise pathogenic mechanism which renders the HTT protein functional with a polyQ tract less than 35 glutamines, but devastatingly damaging above a threshold of 40 glutamines, and which HTT species give rise to this breach in polyQ threshold, remains a fundamental yet unanswered question in the HD field.…”